Method for Treating Multiple Sclerosis

ABSTRACT

A method for treating a multiple sclerosis is provided. The method includes administering a composition containing a plurality of mesenchymal stem cells to a subject in need for a treatment of the multiple sclerosis, in which each of the mesenchymal stem cells expresses insulin-like growth factor 1 receptors.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 15/853,989, filed Dec. 26, 2017, which is a DivisionalApplication of the application Ser. No. 14/934,162, filed Nov. 6, 2015,and claims priority to Taiwan Application Serial Number 104123719, filedJul. 22, 2015, and this application claims priority to China ApplicationSerial Number 201811312204.3, filed Nov. 6, 2018, all of which areherein incorporated by reference.

BACKGROUND Field of Invention

The present disclosure relates to a method for treating a multiplesclerosis. More particularly, the present disclosure relates to a methodfor treating a multiple sclerosis by using mesenchymal stem cellsexpressing a special receptor.

Description of Related Art

Stem cells are undifferentiated biological cells that have abilities toduplicate and self-renew for long periods of time and differentiate intomature cells with specialized cell type and function. The stem cells canbe classified into embryonic stem cells (ESCs) and adult stem cellsaccording to their origin. The ESCs can be obtained from an inner cellmass of a blastocyst, and the adult stem cells can be obtained fromvarious tissues. The stem cells can be further classified intototipotent stem cells, pluripotent stem cells, and multipotent stemcells according to their pluripotent ability. The totipotent stem cellshave a full differentiation capability to develop into a complete embryoor an organism. The pluripotent stem cells have the potential todifferentiate into three germ layers and then differentiate into almostany cells in a tissue or an organ, but the pluripotent stem cells areunable to develop into the complete embryo or the organism. Themultipotent stem cells are the stem cells of specialized tissues, suchas neural stem cells, hematopoietic stem cells, hepatic stem cells, andepidermal stem cells.

Mesenchymal stem cells, a kind of the adult stem cells, belong tomultipotent stem cells. Therefore, the mesenchymal stem cells arecapable of proliferation and multipotent differentiation; that is, theycan differentiate into a variety of mesenchymal tissues such asneurocytes, vascular cells, glial cells, adipocytes or osteocytes. Themesenchymal stem cells can be obtained from the mesenchymal tissues of abone marrow, an adipose, a dental pulp or an umbilical cord. Themesenchymal stem cells have a tendency ability to differentiate intospecific tissues based on their origins of the tissues. Furthermore,when body tissues damage; the mesenchymal stem cells can proceed torepair the damaged tissue directly or indirectly.

The mesenchymal stem cells can be applied for repairing damage of anerve, a heart, a liver, a lung, a kidney, a bone, a cartilage and aretinal. In recent years, it is also found that the mesenchymal stemcells have the capability of an immune adjustment. As such, themesenchymal stem cells are potentially used for treating abnormal immunediseases. In addition, since the mesenchymal stem cells have lowerantigenicity than that of other stem cells, accurate matching is notrequired for the mesenchymal stem cells before transplantation, unlikethat in the clinical applications of the hematopoietic stem cells. Also,the applications of the mesenchymal stem cells have no ethical concernsas that in the use of ESCs. Therefore, the mesenchymal stem cells are anideal source of cell treatment. In the clinical applications, thecapabilities of self-renew and multipotent differentiation of themesenchymal stem cells are essential. Accordingly, cell surfacereceptors associated with pluripotency maintain of the mesenchymal stemcells become one of the main topics in the stem cell medical technologyresearch and development.

SUMMARY

According to one aspect of the present disclosure, a method for treatinga multiple sclerosis is provided. The method includes administering acomposition containing a plurality of mesenchymal stem cells to asubject in need for a treatment of the multiple sclerosis, wherein themesenchymal stem cell expresses insulin-like growth factor 1 receptors.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by Office upon request and payment ofthe necessary fee. The present disclosure can be more fully understoodby reading the following detailed description of the embodiment, withreference made to the accompanying drawings as follows:

FIG. 1A is a micrograph of a primary cell culture of umbilicalmesenchymal stem cells (UMSCs).

FIG. 1B shows analytical results of insulin-like growth factor 1receptor (IGF1R) expressions of mesenchymal stem cells (MSCs) accordingto one embodiment of the present disclosure.

FIG. 10 shows the analytical result of interleukin 22 receptor alpha 1(IL22RA1) expressions of the MSCs according to one embodiment of thepresent disclosure.

FIGS. 1D and 1E is a set of histograms of FACS (fluorescence-activatedcell sorting) analysis showing Wharton's jelly specific cell surfacemolecules expressions of the MSCs according to one embodiment of thepresent disclosure.

FIG. 1F shows analytical results of pluripotent markers expressions ofthe MSCs according to one embodiment of the present disclosure.

FIG. 2 is an exponential growth curve of the MSCs according to oneembodiment of the present disclosure.

FIG. 3A shows analytical results of human cytokines arrays of a humancord blood serum (hUCS) and a fetal calf serum (FCS).

FIG. 3B shows quantitative results of enzyme-linked immunosorbent assay(ELISA) of the hUCS and the FCS.

FIG. 4A shows analytical results of the IGF1R expressions of the MSCsafter transducing shRNA according to one embodiment of the presentdisclosure.

FIG. 4B is an exponential growth curve of the MSCs according to oneembodiment of the present disclosure.

FIG. 4C is quantitative results of BrdU chemiluminescence ELISAs of theMSCs according to one embodiment of the present disclosure.

FIG. 5A is a set of histograms of FACS double staining analysis of theMSCs according to one embodiment of the present disclosure.

FIG. 5B is a micrograph of an immunofluorescence double staining assayof the MSCs according to one embodiment of the present disclosure.

FIG. 5C shows quantitative real-time polymerase chain reaction (qRT-PCR)results of the pluripotent markers of the MSCs according to oneembodiment of the present disclosure.

FIG. 6A is a micrograph showing that the MSCs differentiate intodifferent tissue cells according to one embodiment of the presentdisclosure.

FIG. 6B is a micrograph showing that the MSCs differentiate intoneuroglial cells according to one embodiment of the present disclosure.

FIG. 7A shows the analytical results of the IGF1R expression and a C-X-Cchemokine receptor type 4 (CXCR4) expression of the MSCs after treatingdifferent dose of insulin-like growth factor 1 (IGF1) according to oneembodiment of the present disclosure.

FIG. 7B shows the analytical results of the IGF1R expression and theCXCR4 expression of the MSCs after treating different dose ofplatelet-derived growth factor BB (PDGF-BB) according to one embodimentof the present disclosure.

FIG. 7C shows analytical results of the IGF1R expression and the CXCR4expression of the MSCs after treating different dose of the IGF1 and thePDGF-BB simultaneously according to one embodiment of the presentdisclosure.

FIG. 7D shows analytical results of a phospho-Akt (p-Akt) expression anda phospho-Stat3 (p-Stat3) expression of the MSCs after treatingdifferent dose of the IGF1 and the PDGF-BB simultaneously according toone embodiment of the present disclosure.

FIG. 8A shows analytical results of a body swing test of the rats.

FIG. 8B shows analytical results of the body swing test of the ratsperformed a blocking experiment.

FIG. 9A shows analytical results of a vertical activity in a locomotoractivity test.

FIG. 9B shows analytical results of a vertical movement time in thelocomotor activity test.

FIG. 9C shows analytical results of a number of the vertical movementsin the locomotor activity test.

FIG. 10 shows analytical results of a grip strength test of the rats.

FIG. 11A is a [¹⁸F] fluoro-2-deoxyglucose positron emission tomography(FDG-PET) image of the rats administered with a cell treatment.

FIG. 11B is a quantitative diagram of the [¹⁸F] FDG-PET image of therats administered with the cell treatment.

FIG. 12 shows analytical results of anti-apoptotic proteins expressionlevels of an ischemic area in the rats administered with the celltreatment.

FIG. 13A is a micrograph showing that bisbenzimide and human nuclearantigen (hNA) are co-localization in the ischemic area of the ratsadministered with the cell treatment.

FIG. 13B is a micrograph showing number of implanted IGF1R⁺ mesenchymalstem cells (IGF1R⁺ MSCs) in brain tissues of the rats administered withthe cell treatment.

FIG. 14A is a micrograph showing that glial fibrillary acidic protein(GFAP) is co-localized with the IGF1R or the CXCR4 in the IGF1R⁺ MSCswhich are implanted into the ischemic area of stroke-induced rats.

FIG. 14B is a micrograph showing that microtubule-associated protein 2(MAP-2) is co-localized with the IGF1R or the CXCR4 in the IGF1R⁺ MSCswhich are implanted into the ischemic area of the stroke-induced rats.

FIG. 14C is a micrograph showing that neuronal nuclear antigen (NeuN) isco-localized with the IGF1R or the CXCR4 in the IGF1R⁺ MSCs which areimplanted into an ischemic area of the stroke-induced rats.

FIG. 15A is a micrograph showing that Von Willebrand factor (vWF) isco-localized with the IGF1R or the CXCR4 in the IGF1R⁺ MSCs which areimplanted into the ischemic area of the stroke-induced rats.

FIG. 15B shows analytical results of a FITC-dextran perfusion study inthe stroke-induced rats administered with the IGF1R⁺ MSCs treatment.

FIG. 15C shows analytical results of blood vessel density assays in thestroke-induced rats administered with the IGF1R⁺ MSCs treatment.

FIG. 16 shows analytical results of cerebral blood flow (CBF) in thestroke-induced rats administered with the IGF1R⁺ MSCs treatment.

FIG. 17A shows analytical results of in vivo neurite regeneration test.

FIG. 17B shows analytical results of in vitro neurite regeneration test.

FIG. 18 is a photograph showing a myocardial infarction (MI) area inacute myocardial infarction (AMI) induced rats treated with the IGF1R⁺MSCs.

FIG. 19A is a transthoracic echocardiogram of the AMI-induced ratstreated with the IGF1R⁺ MSCs.

FIG. 19B is a quantitative diagram of the transthoracic echocardiogramof the AMI-induced rats treated with the IGF1R⁺ MSCs.

FIG. 20 shows analytical results of an immunohistochemical analysis inthe AMI-induced rats at 3 days after the IGF1R⁺ MSCs treatment.

FIG. 21 shows analytical results of the immunofluorescence stainingassay in the AMI-induced rats at 3 days after the IGF1R⁺ MSCs treatment.

FIG. 22 shows analytical results of the expressions of pro-inflammatoryfactors in the AMI-induced rats at 3 days after the IGF1R⁺ MSCstreatment.

FIG. 23 shows analytical results of a Masson's trichrome stain in theAMI-induced rats at 28 days after the IGF1R⁺ MSCs treatment.

FIG. 24 is a graph showing the improvement of neurological behavior inexperimental autoimmune encephalitis-induced mice after the celltreatment.

DETAILED DESCRIPTION

A mesenchymal stem cell (MSC) expressing a special cell receptor isprovided. The MSC has a self-renewal capability and a multipotentcapability. A method for a clonogenic expansion of a plurality ofmesenchymal stem cells (MSCs) is also provided. The method can enhance aspecial cell receptor expression of the MSCs and maintain themultipotent capability of the MSCs. A method for obtaining a pluralityof multipotent MSCs is further provided. The method can quickly andspecifically screen the MSCs having the multipotent capability from amammalian tissue cell mixture. Furthermore, uses of the mesenchymal stemcell are provided for treating an ischemic heart disease and a braintissue damage.

In more details, aforementioned MSC expresses an insulin-like growthfactor 1 receptor (IGF1R). The term “IGF1R⁺ mesenchymal stem cell(IGF1R⁺ MSCs)” is used in the specification to represent the MSC of thepresent disclosure. The IGF1R⁺ MSCs are multipotent. The IGF1R⁺ MSCs canbe human cells; especially the IGF1R⁺ MSCs can be umbilical cordmesenchymal stem cells (UMSCs). The method for the clonogenic expansionof a plurality of MSCs includes culturing the IGF1R⁺ MSCs in mediacontaining a human cord blood serum (hUCS). A concentration of the hUCSis 1-10% (v/v); preferably, the concentration of the hUCS is 2% (v/v).The method for obtaining a plurality of the multipotent MSCs includesscreening IGF1R positive cells from the mammalian tissue cell mixture toobtain the multipotent MSCs. The method for obtaining a plurality of themultipotent MSCs can further screen interleukin 22 receptor alpha 1(IL22RA1) positive cells from the mammalian tissue cell mixture toobtain the multipotent MSCs. The mammalian tissue can be selected fromthe group consisting of a bone marrow, a dental pulp, a placenta, anumbilical cord, an adipose tissue, and combinations thereof.

The IGF1R⁺ MSCs can be used for treating the ischemic heart disease andthe brain tissue damage. In more details, the IGF1R⁺ MSCs can attenuatepost-myocardial infarction (MI) left ventricle (LV) dysfunction, reducean infarct size after the MI, reduce a fibrosis caused by the MI, andreduce an inflammatory effect on the MI. Therefore, the IGF1R⁺ MSCs cantreat the ischemic heart disease, wherein the ischemic heart disease canbe the MI. Furthermore, the IGF1R⁺ MSCs can increase a glucose metabolicactivity of the subject, enhance an angiogenesis of the subject, andaugments a neurite regeneration of the subject. The IGF1R⁺ MSCs have aneuroplasticity effect through IGF1R and C-X-C chemokine receptor type 4(CXCR4) interactions. Therefore, the IGF1R⁺ MSCs can treat the braintissue damage, wherein the brain tissue damage can be a cerebralischemic disease (such as a stroke) or a neural degenerative disease(such as a Parkinson's disease).

The following are descriptions of the specific terms used in thespecification:

The term “IGF1R (insulin-like growth factor 1 receptor)” means a proteinfound on the surface of human cells, and the IGF1R is a transmembranereceptor that is activated by a hormone called insulin-like growthfactor 1 (IGF1). The IGF1R belongs to the large class of tyrosine kinasereceptors. The IGF1 is a polypeptide protein hormone similar inmolecular structure to insulin. In addition, the IGF1 plays an importantrole in growths and anabolism of adults. The term “IL22RA1 (interleukin22 receptor alpha 1)” means a protein shown to be a receptor forinterleukin 22 (IL22). The IL22 is a cytokine having bothanti-inflammatory and pro-inflammatory effects, and the IL22 is secretedby a variety of immune cells.

The term “CXCR4 (C-X-C chemokine receptor type 4)” means analpha-chemokine receptor specific for stromal-derived-factor-1 (SDF-1),a molecule endowed with a potent chemotactic activity for lymphocytes.The CXCR4 is expressed on most body tissues and organs. The CXCR4 is a Gprotein-coupled receptor (GPCR) composed of 352 amino acids, and theCXCR4 has seven transmembrane structures.

Reference will now be made in detail to the present embodiments of thepresent disclosure, examples of which are illustrated in theaccompanying drawings. Wherever possible, the same reference numbers areused in the drawings and the description to refer to the same or likeparts.

Examples I. The IGF1R⁺ MSCs 1.1 Preparation of the IGF1R⁺ MSCs

To prepare the IGF1R⁺ MSCs, the mammalian tissues used in this exampleare human umbilical cord tissues. The UMSCs of the human umbilical cordtissue are stored in Wharton's jellies. The collected human umbilicalcord tissues are washed three times with Ca2⁺ and Mg2⁺-free PBS (DPBS,Life Technology). The human umbilical cord tissues are mechanically cutby scissors in a midline direction and the vessels of the umbilicalartery, vein and outlining membrane are dissociated from the Wharton'sjelly. The Wharton's jelly is then extensively cut into pieces smallerthan 0.5 cm³, treated with collagenase type 1 (Sigma), and thenincubated for 14-18 h at 37° C. in a 95% air/5% CO₂ humidifiedatmosphere. Explants then are cultured in DMEM containing 2% hUCS or 10%fetal calf serum (FCS) and antibiotics at 37° C. in a 95% air/5% CO₂humidified atmosphere. They are left undisturbed for 5-7 days to allowfor migration of the cells from the explants.

FIG. 1A is a micrograph of primary cell culture of the UMSCs, whereinthe black arrow indicates the explant. In FIG. 1A, the cells migratefrom the explants, and a cellular morphology of the cells becomeshomogenously spindle shaped in cultures after 4-8 passages.

The specific surface molecules of the cells from the Wharton's jelly arecharacterized by a flow cytometric analysis. The specific surfacemolecules detected in this example are the IGF1R, the IL22RA1, Wharton'sjelly specific cell surface molecules, and pluripotent markers. TheWharton's jelly specific cell surface molecules include CD13, CD29,CD34, CD44, CD45, CD73, CD90, CD105, CD117, CD166, HLA-ABC, and HLA-DR.The pluripotent markers include Oct-4, Sox-2, Nanog, and SSEA-4.

FIG. 1B shows analytical results of the IGF1R expressions of the IGF1R⁺MSCs. FIG. 10 shows the analytical result of interleukin 22 receptoralpha 1 (IL22RA1) expressions of the IGF1R⁺ MSCs. FIGS. 1D and 1E is aset of histograms of FACS (fluorescence-activated cell sorting) analysisshowing Wharton's jelly specific cell surface molecules expressions ofthe IGF1R⁺ MSCs. FIG. 1F shows the analytical results of pluripotentmarkers expressions of the IGF1R⁺ MSCs.

In FIG. 1B, we first detect the IGF1R expression on the cells from theWharton's jelly by the flow cytometric analysis, and then detect theIGF1R expression on the MSCs form different tissues by a Westernblotting analysis. The MSCs analyzed in the Western blotting analysisare human fibroblasts, human decidua-derived stem cells (hDSCs), humanadipose derived stem cells (ADSCs), the UMSCs, and human bone marrowstromal cell (BMSC). Based on the analytical results, the cells from theWharton's jelly (UMSCs) express the surface markers of the IGF1R, whichshows consensus expression in the BMSCs, ADMSCs, and hDSCs.

In FIGS. 1C-1F, IL22RA1, Wharton's jelly specific cell surface moleculesand pluripotent markers of the cells from the Wharton's jelly arecharacterized by the flow cytometric analysis. In FIG. 10, the cellsfrom the Wharton's jelly express the IL22RA1, and 45.4% cells expressthe IL22RA1 compared to a control group. In FIGS. 1D and 1E, the cellsfrom the Wharton's jelly express CD13, CD29, CD44, CD73, CD90, CD105,CD166, and HLA-ABC. In addition, the cells from the Wharton's jelly donot express CD34, CD45, CD 117, and HLA-DR. The flow cytometric analysisresults indicate that the cells from the Wharton's jelly are a penotypeof the MSCs rather than hematopoietic stem cells. In FIG. 1F, the cellsfrom the Wharton's jelly express Oct-4, Sox-2, Nanog, and SSEA-4; thoseare the pluripotent markers represented the multipotent capability.

Thereafter, the cells are analyzed using a flow cytometer (BectonDickinson). Cells sorting is used to purify the IGF1R⁺ MSCs (>95% IGF1R)using a FACSTAR⁺ flow-cytometer (Becton Dickinson). Approximately 96% ofthe sorted cells are viable, as confirmed by Trypan blue exclusion test.

1.2 Culture of the IGF1R⁺ MSCs

The sorted IGF1R⁺ MSCs are cultured in the DMEM containing 2% hUCS or10% fetal calf serum (FCS) and antibiotics at 37° C. in a 95% air/5% CO₂humidified atmosphere. The IGF1R⁺ MSCs cultured in 2% hUCS and in 10%FCS are analyzed their growth kinetics.

FIG. 2 is an exponential growth curve of the IGF1R⁺ MSCs. In FIG. 2, thehUCS-cultured IGF1R⁺ MSCs (U-IGF1R⁺ MSCs) proliferate faster compared toFCS-cultured IGF1R⁺ MSCs (F-IGF1R⁺ MSCs). The U-IGF1R⁺ MSCs growexponentially with a doubling time of 22 hours and are extensivelyexpanded for more than 150 days without signs of senescence andspontaneous differentiation.

To assess the advantages of using the medium containing the hUCS toculture the IGF1R⁺ MSCs, we apply a human cytokine array system(RayBiotech) to identify and compare the specific cytokine(s) in thehUCS and the FCS. A total of 42 cytokines are examined in this example,and cytokine expression levels in the hUCS or the FCS are determined bya densitometry.

FIG. 3A shows the analytical results of human cytokines arrays of thehUCS and the FCS, wherein “P” represents a positive control and “N”represents a negative control. In FIG. 3A, expressions levels of fivecytokines are significantly higher in the hUCS than in the FCS. Theseare epidermal growth factor (EGF, square 1), angiogenin (ANG, square 2),macrophage inflammatory protein (MIP-1δ, square 3), regulated onactivation, normal T-cell expressed and presumably secreted (RANTES,square 4) and platelet-derived growth factor BB (PDGF-BB). The ratio ofdifferences expression levels of these five cytokines are 2, 3, 3, 2,and 4 times, respectively. In contrast, expression levels of the IGF1are similar in the hUCS and the FCS.

To more accurately quantify the PDGF-BB and the IGF1 concentration inthe hUCS and the FCS, enzyme-linked immunosorbent assay (ELISA) isperformed using the hUCS and the FCS samples in this example to analyzethe PDGF-BB concentration and the IGF1 concentration.

FIG. 3B shows quantitative results of ELISA of the hUCS and the FCS. InFIG. 3B, the IGF1 expression levels are indistinguishable between thetwo types of serum, while the PDGF-BB concentration is substantiallyhigher in the hUCS than in the FCS (p<0.05).

1.3 the Self-Renewal Capability of the IGF1R⁺ MSCs

To investigate whether the IGF1R signaling pathway contributes to theregulation of the IGF1R⁺ MSCs self-renewal capability, we uselentivirus-mediated shRNA targeting of IGFR1R (LV-IGF1R-sh,sc-29358-V,Santa Cruz Biotechnology) to the hUCS-cultured IGF1R⁺ MSCs (U-IGF1R⁺MSCs) or the FCS-cultured IGF1R⁺ MSCs (F-IGF1R⁺ MSCs) for knocking downlevels of the receptor. We also use lentivirus-mediated control shRNA(LV-control-sh, Santa Cruz Biotechnology) to the U-IGF1R⁺ MSCs or theF-IGF1R⁺ MSCs as the control group. After 48 hours lentivirus infection,the IGF1R protein expressions of the infected-IGF1R⁺ MSCs are detectedby the Western blotting analysis. The infected-IGF1R⁺ MSCs are alsoanalyzed their growth kinetics.

FIG. 4A shows the analytical results of the IGF1R expressions of theIGF1R⁺ MSCs after transducing shRNA. FIG. 4B is the exponential growthcurve of the IGF1R⁺ MSCs. There are four groups in FIG. 4A, the U-IGF1R⁺MSCs transduced with or without LV-IGF1R-sh or the U-IGF1R⁺ MSCstransduced with or without LV-control-sh, wherein all U-IGF1R⁺ MSCs inFIG. 4A are cultured in the medium containing the hUCS. In FIG. 4A, thegroup of the IGF1R⁺ MSCs transduced with LV-IGF1R-sh exhibits asignificant reduction in the IGF1R protein expression 48 hourspost-infection compared with the group transduced with LV-control-sh,while the IGF1R pretein expression are equivalent in other groups. Thereare four groups in FIG. 4B, the IGF1R⁺ MSCs cultured in the mediumcontaining the hUCS and transduced with the LV-control-sh (LV-control-shU-IGF1R⁺ MSCs), the IGF1R⁺ MSCs cultured in the medium containing theFCS and transduced with the LV-control-sh (LV-control-sh F-IGF1R⁺ MSCs),the IGF1R⁺ MSCs cultured in the medium containing the hUCS andtransduced with the LV-IGF1R-sh (LV-IGF1R-sh U-IGF1R⁺ MSCs), and theIGF1R⁺ MSCs cultured in the medium containing the FCS and transducedwith the LV-IGF1R-sh (LV-IGF1R-sh F-IGF1R⁺ MSCs), respectively. In FIG.4B, the growth kinetic shows that the LV-IGF1R-sh U-IGF1R⁺ MSCs and theLV-IGF1R-sh F-IGF1R⁺ MSCs proliferate more slowly than the LV-control-shU-IGF1R⁺ MSCs and the LV-control-sh F-IGF1R⁺ MSCs. It indicates that theLV-IGF1R-sh transduction reduces the proliferation of the IGF1R⁺ MSCs.

To further evaluate the IGF1R⁺ MSCs proliferation potential, we useBromodeoxyuridine (BrdU) to label DNA and perform BrdU chemiluminescenceELISAs in this example. After a 4-6 hours starvation (incubation in themedium lacking supplements), the IGF1R⁺ MSCs are incubated in the mediumcontaining 10% FCS or 2% hUCS with supplement or the SDF-1α (100 ng/mL,positive control) for 2 days and then transduced with the LV-control-shor the LV-IGF1R-sh. The proliferation of the IGF1R⁺ MSCs are tested bymeasuring BrdU incorporation using a BrdU chemiluminescence immunoassaykits (Roche), and further confirmed by counting Trypan blue cell.

FIG. 4C is the quantitative results of BrdU chemiluminescence ELISAs ofthe IGF1R⁺ MSCs. There are five groups in FIG. 4C, the IGF1R⁺ MSCscultured in the medium containing the SDF-1α as the positive control,the IGF1R⁺ MSCs cultured in the medium containing the hUCS andtransduced with the LV-control-sh, the IGF1R⁺ MSCs cultured in themedium containing the FCS and transduced with the LV-control-sh, theIGF1R⁺ MSCs cultured in the medium containing the hUCS and transducedwith the LV-IGF1R-sh, and the IGF1R⁺ MSCs cultured in the mediumcontaining the FCS and transduced with the LV-IGF1R-sh, respectively. InFIG. 4C, the IGF1R⁺ MSCs cultured in the hUCS show significant higherBrdU incorporation compared to the IGF1R⁺ MSCs cultured in the FCS. Incontrast, the LV-IGF1R-sh transductions abolish the BrdU incorporationin the IGF1R⁺ MSCs cultured in either the hUCS or the FCS. It indicatesthat the IGF1R is essential for the IGF1R⁺ MSCs proliferation.

1.4 the Multipotent Capability of the IGF1R⁺ MSCs

To investigate whether the multipotent capability of the IGF1R⁺ MSCs isrelated to the IGF1R, we perform FACS double staining analysis and animmunofluorescent double staining assay. The IGF1R⁺ MSCs are stainedwith IGF1R/Oct-4 antibodies, IGF1R/Sox-2 antibodies, IGF1R/Nanogantibodies, and IGF1R/SSEA4 antibodies, respectively. The stained IGF1R⁺MSCs are measured co-expressions between the IGF1R and the pluripotentmarkers using the flow cytometer and the immunofluorescent doublestaining assay.

FIG. 5A is a set of histograms of FACS double staining analysis of theIGF1R⁺ MSCs. FIG. 5B is a micrograph of Immunofluorescence doublestaining assay of the IGF1R⁺ MSCs, wherein the IGF1R⁺ MSCs are obtainedfrom five independent samples. In FIGS. 5A and 5B, the results revealthat the IGF1R is co-expressed with the pluripotent markers of Oct-4,Sox-2, Nanog and SSEA-4. In FIG. 5B, the results further reveal that theIGF1R is co-expressed with the CXCR4.

We perform quantitative real-time polymerase chain reaction (qRT-PCR) tofurther investigate the difference in the pluripotent markersexpressions between the IGF1R⁺ MSCs and IGF1R⁻ MSCs (the mesenchymalstem cells do not express IGF1R). FIG. 5C shows qRT-PCR results of thepluripotent markers of the MSCs, wherein the test cells are the IGF1R⁺MSCs, the IGF1R⁻ MSCs, human fibroblasts as the negative control, andinduced pluripotent stem cells (iPS) as the positive control. In FIG.5C, the IGF1R⁺ MSCs express higher levels of Oct-4, Sox-2, Nanog andSSEA-4 than that in the IGF1R⁻ MSCs. It indicates that the IGF1R isessential for the IGF1R⁺ MSCs maintaining the multipotent capability.

1.5 In Vitro Differentiation of the IGF1R⁺ MSCs

To investigate whether the IGF1R⁺ MSCs cultured in the medium containingthe hUCS or the FCS could affect the multipotent capability of theIGF1R⁺ MSCs, we perform the in vitro differentiation assay in thisexample. The fifth to tenth-passaged IGF1R⁺ MSCs (cultured in the mediumcontaining the hUCS or the FCS) are seeded at a density of 5×10³cells/cm² in cell culture dishes and then cultured in differentdifferentiation medium to induce an adipogenic differentiation, achondrogenic differentiation, an osteogenic differentiation, a vasculartubes formation, and a neural cell differentiation. Cell morphologies ofdifferentiated cells are observed by a microscope, and the cell types ofthe differentiated cells are further confirmed by staining assay.

FIG. 6A is the micrograph showing that the IGF1R⁺ MSCs differentiateinto different tissue cells. FIG. 6B is the micrograph showing that theIGF1R⁺ MSCs differentiate into neuroglial cells.

In FIG. 6A, the adipogenic differentiation is confirmed by Oil red Ostain, the osteogenic differentiation is confirmed by Alizarin red Sstain, the chondrogenic differentiation is confirmed by Alcion bluestain, and the vascular tubes formation of the IGF1R⁺ MSCs is confirmedby the cell morphology observation in bright field. The results showthat IGF1R⁺ MSCs have the abilities to differentiate into adipocytes,chondrocytes, and osteocytes and form vascular tubes.

In FIG. 6B, regarding neural differentiation, some IGF1R⁺ MSCs culturedin neural differentiation medium in the dish exhibit refractile cellbody morphology with extended neurite-like cellular processes arrangedinto a network. We further identify mature neural markers expressions ofthe neural differentiated cell using an immunofluorescent assay. Themature neural markers includes glial fibrillary acidic protein (GFAP),microtubule-associated protein 2 (MAP-2), 04 and Neuron-specific classIII beta-tubulin (Tuj-1). In FIG. 6B, IGF1R⁺ MSC-derived neuroglialcells express GFAP, MAP-2, 04 and Tuj-1. It indicates that the IGF1R⁺MSC-derived neuroglial cells are indeed neuroglial cells.

The percentages of four mature neural markers expressions of the IGF1R⁺MSCs cultured in the medium containing 2% hUCS or 10% FCS are shown inTable 1 as follows. In Table 1, the percentages of four mature neuralmarkers expressions are higher in U-IGF1R⁺ MSCs than that in F-IGF1R⁺MSCs. It indicates that the medium containing the hUCS improves theIGF1R⁺ MSCs differentiating into neuroglial cells.

TABLE 1 serum GFAP MAP-2 O4 Tuj-1 2% hUCS 15.2 ± 3.1% 12.1 ± 3.1% 9.4 ±2.1% 10.2 ± 1.7% 10% FCS  8.6 ± 2.2%  7.1 ± 2.7% 5.8 ± 1.6%  6.1 ± 1.5%

1.6 Regulatory Mechanism of the IGF1R⁺ MSCs

To investigate whether the IGF1 alone or with the PDGF-BB (a keyingredient of the hUCS) regulate the IGF1R or the CXCR4 levels of theIGF1R⁺ MSCs, we performed the Western blotting analysis for the IGF1R⁺MSCs. The IGF1R⁺ MSCs are treated with different dose of the IGF1 ordifferent dose of the PDGF-BB, and the protein expressions of the IGF1Rand the CXCR4 are detected by the Western blotting analysis.

FIG. 7A shows the analytical results of the IGF1R expression and theCXCR4 expression of the IGF1R⁺ MSCs after treating different dose ofinsulin-like growth factor 1 (IGF1). FIG. 7B shows the analyticalresults of the IGF1R expression and the CXCR4 expression of the IGF1R⁺MSCs after treating different dose of the PDGF-BB. FIG. 7C shows theanalytical results of the IGF1R expression and the CXCR4 expression ofthe IGF1R⁺ MSCs after treating different dose of the IGF1 and thePDGF-BB simultaneously, wherein * represents p<0.05 and ** representsp<0.01 compared to the control group (the IGF1R⁺ MSCs untreated with theIGF1 and the PDGF-BB). In FIG. 7A, IGF1 treatments reduce expression ofIGF1R in the IGF1R⁺ MSCs in a dose-dependent manner, but the IGF1treatments up-regulate the CXCR4 levels. In particular, the CXCR4 levelis significantly increased by treating 5 nM IGF1. In FIG. 7B, thePDGF-BB dose-dependently increases both the IGF1R and the CXCR4expression in the IGF1R⁺ MSCs. In FIG. 7C, an IGF1-induceddown-regulation of IGF1R is effectively inhibited by addition of thePDGF-BB.

To further determine whether the PDGF-BB is more effective than the IGF1in activating signaling pathways that control cell proliferations, theIGF1R⁺ MSCs are treated with the IGF1 and/or the PDGF-BB (underserum-free conditions) and the expression level of phosphorylated formsof Akt (p-Akt) and Stat3 (p-Stat3) are determined by the Westernblotting analysis.

FIG. 7D shows the analytical results of the p-Akt expression and thep-Stat3 expression of the IGF1R⁺ MSCs after treating different dose ofthe IGF1 and the PDGF-BB simultaneously, wherein * represents p<0.05compared to the control group (the IGF1R⁺ MSCs untreated with the IGF1and the PDGF-BB). In FIG. 7D, PDGF-BB treatments significantlyup-regulate the p-Akt expression and the p-Stat3 expression, whereas theIGF1 treatments have no effect on the p-Akt expression and the p-Stat3expression (either alone or in combination with the PDGF-BB). The IGF1R⁺MSCs are further treated with specific pharmacological inhibitors of thep-Akt (LY294002) and the p-Stat3 (AG490) respectively. PDGF-BB-inducedphosphorylations of the Akt and the Stat3 are completely inhibited byLY294002 and AG490 respectively (data not shown). Taken together, theseresults reveal that the PDGF-BB is more effective than the IGF1 inactivating these important downstream signaling pathways, suggestingthat the hUCS is better for culturing the IGF1R⁺ MSCs than the FCS.

II. The IGF1R⁺ MSCs Used for Treating the Brain Tissue Damage

The data of the first part examples demonstrate that the IGF1R⁺ MSCshave self-renewal capability and multipotent capability. In the secondpart examples, we further discuss the effect on the IGF1R⁺ MSCs used fortreating the brain tissue damage.

2.1 the IGF1R⁺ MSCs Transplantation Improved Neurological Behavior inStroke Rat Model

In vivo self-renewal and neuroregenerative potentials of the IGF1R⁺ MSCsare assessed in a stroke model in the second part examples. Rats beforeand after the stroke are measured by three modalities of neurologicaldeficits to evaluate the neurological recovery.

An ischemia-reperfusion model is used to simulate transient focalcerebral ischemia in rats. Test animals are male Sprague-Dawley (SD)rats weighing 225-275 g. The ischemia-reperfusion model is induced byligations of bilateral common carotid arteries (CCAs) and a right middlecerebral artery (MCA). The bilateral CCAs are clamped with non-traumaticarterial clips, and the right MCA is ligated with an 10-0 nylon suture.After 90 minutes ischemia, the suture on the MCA and the arterial clipson CCAs are removed to allow reperfusion. One hour after brain ischemia,the rats are injected intravenously with approximately 2×10⁶ cells intofemoral vein. The rats are subdivided into five treatment groups: (1) aU-IGF1R⁺ MSCs treatment group (n=8), (2) the IGF1R⁺ MSCs cultured in themedium containing the FCS (F-IGF1R⁺ MSCs treatment group; n=8), (3) theMSCs cultured in the medium containing the hUCS (U-MSCs treatment group;n=8), (4) the MSCs cultured in the medium containing the FCS (F-MSCstreatment group; n=8), and (5) vehicle-control group (n=8). The rats ofthe U-IGF1R⁺ MSCs treatment group further perform a blocking experimentby administering a CXCR4 antibody (CXCR4-Ab mAb 173, R&D System) and anIGF1R inhibitor (PPP, Santa Cruz Biotechnology) to block the CXCR4 andthe IGF1R. For the blocking experiment, the rats of group 1 areadministered the CXCR4-Ab mAb by intraperitoneal injection twice weeklyfor two weeks and received i.p. injections of the PPP (20 mg/kg/day) forthree days.

Neurological behavioral assessments are performed 5 days before cerebralischemia/reperfusion, and 1, 7, 14 and 28 days after celltransplantation. The three modalities of neurological deficits measurebody asymmetry, locomotor activity and grip strength of the rats.

i. A Body Swing Test

The body swing test is used to assess body asymmetry after MCA ligation.Initially, the rats are suspended by their tail 10 cm above the cagefloor, and lateral body movements are recorded. Specifically, thefrequency with which the initial head swing contra-lateral to theischemic side is counted in twenty consecutive tests and is normalizedto the baseline score.

FIG. 8A shows the analytical results of the body swing test of the rats.FIG. 8B shows the analytical results of the body swing test of the ratsperformed a blocking experiment. In FIG. 8A, all rats treated with theMSCs (four MSCs in this example) exhibit significantly better functionalrecovery than control. The neurological recovery of the rats treatedwith the MSCs cultured in the medium containing the hUCS (group 1 andgroup 3) are better than the neurological recovery of the rats treatedwith the MSCs cultured in the medium containing the FCS (group 2 andgroup 4). In particular, the rats treated with the U-IGF1R⁺ MSCs(group 1) show significantly more improvement than all other groups. InFIG. 8B, when the group 1 is treated with the PPP (n=8) or the CXCR4-AbmAb 173 (n=8), the neurological recovery for the body swing test issimilar to control. It indicates that the effect on the functionalrecovery through the U-IGF1R⁺ MSCs treatment is inhibiting by blockingthe CXCR4 and the IGF1R.

ii. A Locomotor Activity Test

The locomotor activity test is measured for about 2 hours using VersaMaxAnimal Activity Monitoring System (Accuscan Instruments), which contains16 horizontal infrared sensors and 8 vertical infrared sensors. Thevertical sensors are situated 10 cm above the chamber floor and thelocomotor activity is quantified by a number of a beam broken by therat's movement in the chamber. Three vertical-movement parameters aremeasured: (i) vertical activity (ii) vertical time (iii) number ofvertical movements by the manufacturer's instruction.

FIG. 9A shows the analytical results of the vertical activity in thelocomotor activity test. FIG. 9B shows the analytical results of thevertical movement time in the locomotor activity test. FIG. 9C shows theanalytical results of the number of the vertical movements in thelocomotor activity test. In FIGS. 9A-9C, all rats treated with the MSCs(four MSCs in this example) exhibit significantly improvement in thelocomotor activity than the control group, and the rats treated with theU-IGF1R⁺ MSCs show significantly more improvement in the locomotoractivity than the rats treated with the F-IGF1R⁺ MSCs. However, thelocomotor activity of group 1 is similar to the control group when thegroup 1 is treated with the PPP or the CXCR4-Ab. It indicates that theeffect on locomotor activity improvement through the U-IGF1R⁺ MSCstreatment is inhibiting by blocking the CXCR4 and the IGF1R.

iii. A Grip Strength Test

The grip strength is analyzed using Grip Strength Meter (TSE-Systems).In brief, the grip strength of each forelimb of the rat is measuredseparately from the mean of 20 pulls, and the ratio of ipsilateral gripstrength to contralateral grip strength is calculated. In addition, theratio of grip strength post-cell-treatment and pre-cell-treatment isalso calculated, and the changes are presented as a percentage of thepre-cell-treatment value.

FIG. 10 shows the analytical results of the grip strength test of therats. In FIG. 10, the rats treated with the IGF1R⁺ MSCs exhibitsignificantly improvement in the grip strength than the control group,and the rats treated with the U-IGF1R⁺ MSCs show significantly moreimprovement in the grip strength than the rats treated with the F-IGF1R⁺MSCs. However, the grip strength of group 1 is similar to the controlgroup when the group 1 is treated with the PPP or the CXCR4-Ab. Itindicates that the effect on grip strength improvement through theU-IGF1R⁺ MSCs treatment is inhibiting by blocking the CXCR4 and theIGF1R.

These results of three modalities of neurological deficits suggest thatthe IGF1R⁺ MSCs can improve the neurological behaviors in stroke-inducedrats. In particular, the U-IGF1R⁺ MSCs rather than the F-IGF1R⁺ MSCshave superior neuroregenerative potential that required the IGF1R andthe CXCR4 receptor pathways.

2.2 the IGF1R⁺ MSCs Transplantation Enhance a Glucose Metabolism

To investigate whether the U-IGF1R⁺ MSCs implantation affected glucosemetabolic activity following cerebral ischemia, the glucose metabolicactivity of the stroke-induced rats treated with the U-IGF1R⁺ MSCs areexamined by using microPET to perform [¹⁸F] fluoro-2-deoxyglucosepositron emission tomography (FDG-PET).

FIG. 11A is a [¹⁸F] image of the rats administered with a celltreatment. FIG. 11B is a quantitative diagram of the [¹⁸F] FDG-PET imageof the rats administered with the cell treatment. The rats aresubdivided into five cell treatment groups: the U-IGF1R⁺ MSCs treatmentgroup (n=6), the F-IGF1R⁺ MSCs treatment group (n=6), the U-IGF1R⁺ MSCstreatment and the PPP injection simultaneously group (n=6), the U-IGF1R⁺MSCs treatment and the CXCR4-Ab injection simultaneously group (n=6),and the control group (n=6). The rats are performed [¹⁸F] FDG-PET atfour weeks after the cell treatment. In FIG. 11A, the IGF1R⁺ MSCsimplantation increase the FDG uptake in damaged cortical regions (rightside of the brain), and the FDG uptake enhancement in the U-IGF1R⁺ MSCstreatment group is greater than the F-IGF1R⁺ MSCs treatment group andcontrol group. FIG. 11B is a semiquantitative measurement of the [¹⁸F]FDG-PET image. In FIG. 11B, the enhancement in the glucose metabolicactivity is abolished in the U-IGF1R⁺ MSCs treatment and the PPPinjection simultaneously group or the U-IGF1R⁺ MSCs treatment and theCXCR4-Ab injection simultaneously group. It indicates that the effect onthe glucose metabolic activity enhancement through the U-IGF1R⁺ MSCstreatment is inhibiting by blocking the CXCR4 and the IGF1R.

2.3 Intravenous Transplantation of the IGF1R⁺ MSCs Increases theExpression of Anti-Apoptotic Proteins In Vivo

In order to determine whether the stroke-induced rats treated with theU-IGF1R⁺ MSCs exhibit improved neurological function by means ofup-regulation of survival factors, we examine the protein expression ofthe anti-apoptotic proteins in the ischemic area of the stroke-inducedrats using the Western blotting analysis. The detected anti-apoptoticproteins are B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large(Bcl-xL). The expression of pro-apoptotic proteins are also detected inthis example, wherein the detected pro-apoptotic proteins areBCl2-associated X protein (Bax) and BCl2-associated death promoter(Bad).

FIG. 12 shows the analytical results of anti-apoptotic proteinsexpression levels of the ischemic area in the rats administered with thecell treatment. In FIG. 12, the protein expressions of the Bcl-2 and theBcl-xL of the ischemic area are increased in the rats administered withthe IGF1R⁺ MSCs treatment. Significant up-regulations of anti-apoptoticproteins of the Bcl-2 and the Bcl-xL are found in the U-IGF1R⁺ MSCstreatment group (n=6) compared with the F-IGF1R⁺ MSCs treatment group(n=6) and the control group (n=6), wherein p<0.05 when the F-IGF1R⁺ MSCstreatment group compares to the control group, and p<0.01 when theU-IGF1R⁺ MSCs treatment group compares to the control group. Incontrast, the protein expression levels of the Bax and the Bad in theischemic area are not affected by the IGF1R⁺ MSCs treatment (theF-IGF1R⁺ MSCs treatment and the U-IGF1R⁺ MSCs treatment).

2.4 the IGF1R⁺ MSCs Treatment Enhances Neural Differentiation In Vivo

To analyze whether transplanted the IGF1R⁺ MSCs could differentiate intothe neuroglial cells, we perform the immunofluorescence staining assayto determine number of the implanted IGF1R⁺ MSCs in the ischemic area ofthe rats and the immunofluorescence double staining assay to determinethe differentiation cell-type of the implanted IGF1R⁺ MSCs using laserscanning confocal microscopy at 28 days after transplantation.

FIG. 13A is the micrograph showing that bisbenzimide and human nuclearantigen (hNA) are co-localization in the ischemic area of the ratsadministered with the cell treatment. FIG. 13B is the micrograph showingnumber of implanted IGF1R⁺ MSCs in the brain tissues of the ratsadministered with the cell treatment. In FIG. 13A, allbisbenzimide-labeled IGF1R⁺ MSCs express the hNA, confirming their humanorigin. In FIG. 13B, the U-IGF1R⁺ MSCs treatment group (n=8) have moreimplanted IGF1R⁺ MSCs in the ischemic area than the F-IGF1R⁺ MSCstreatment group (n=8) (p<0.05). However, the blocking experiment (theU-IGF1R⁺ MSCs treatment and the PPP injection simultaneously group orthe U-IGF1R⁺ MSCs treatment and the CXCR4-Ab injection simultaneouslygroup) results show that the difference is abolished by the injection ofthe PPP and the CXCR4-Ab.

To further confirm the differentiation cell-type of the implanted IGF1R⁺MSCs, we perform the immunofluorescence double staining assay labelingcell-type specific markers, the IGF1R, and the CXCR4 to determine theco-localization of the cell-type specific markers and the IGF1R, and theco-localization of the cell-type specific markers and the CXCR4. Thenucleus is labeled by the bisbenzimide. The cell-type specific markersinclude glial fibrillary acidic protein (GFAP), microtubule-associatedprotein 2 (MAP-2), and neuronal nuclear antigen (NeuN).

FIG. 14A is the micrograph showing that the GFAP is co-localized withthe IGF1R or the CXCR4 in the IGF1R⁺ MSCs which are implanted into theischemic area of the stroke-induced rats. FIG. 14B is the micrographsshowing that the MAP-2 is co-localized with the IGF1R or the CXCR4 inthe IGF1R⁺ MSCs which are implanted into the ischemic area of thestroke-induced rats. FIG. 14C is the micrographs showing that the NeuNis co-localized with the IGF1R or the CXCR4 in the IGF1R⁺ MSCs which areimplanted into the ischemic area of the stroke-induced rats.

In FIGS. 14A-14C, some bisbenzimide-labeled cells that express the CXCR4are co-localized with neural markers of the GFAP, the MAP-2, and theNeu-N, respectively. In addition, some bisbenzimide-labeled cells thatexpress the IGF1R are co-localized with neural markers of the GFAP, theMAP-2, and the Neu-N, respectively.

The percentages of the co-localization of the bisbenzimide-labeled cellsand the GFAP, the MAP-2, and the Neu-N in the immunofluorescence doublestaining assay are shown in Table 2 as follows, wherein brain tissues ofthe stroke-induced rats implanted the U-IGF1R⁺ MSCs (n=8) or the braintissues of the stroke-induced rats implanted the F-IGF1R⁺ MSCs (n=8) arelabeled by the immunofluorescence double staining assay. In Table 2, thepercentages of the bisbenzimide-labeled cells co-localizing with theGFAP, the MAP-2 and the Neu-N are significantly higher in the U-IGF1R⁺MSCs treated rats (9.5%, 12%, and 10%, respectively) (n=8) than in theF-IGF1R⁺ MSCs treated rats (4%, 5%, and 4%, respectively). It suggestshigher levels of neurogenesis rate in the stroke-induced rats thatreceived the U-IGF1R⁺ MSCs treatment.

TABLE 2 marker Bisbenzimide/ Bisbenzimide/ Bisbenzimide/ Cell type GFAPMAP-2 NeuN U-IGF11R⁺ MSCs 9.5% 12% 10% F-IGF11R⁺ MSCs   4%  5%  4%

2.5 the IGF1R⁺ MSCs Transplantation Promotes an Angiogenesis In Vivo

To determine whether the U-IGF1R⁺ MSCs transplantation potentiatesangiogenesis in the ischemic area of the stroke-induced rats, we performthe immunofluorescence double staining assay, FITC-dextran perfusionstudies, and blood vessel density assays in this example.

Brain slices of the stroke-induced rats are performed theimmunofluorescence double staining assay labeling vascular endothelialcell marker Von Willebrand factor (vWF), the IGF1R and the CXCR4 todetermine the co-localization of the vWF and the IGF1R, and theco-localization of the vWF and the CXCR4. The nucleus is labeled by thebisbenzimide. FIG. 15A is the micrographs showing that vWF isco-localized with the IGF1R or the CXCR4 in the IGF1R⁺ MSCs which areimplanted into the ischemic area of the stroke-induced rats. In FIG.15A, several bisbenzimide-labeled cells co-expressing the IGF1R and theCXCR4 are co-localized with cells of vascular phenotype (vWF⁺) in theperivascular and endothelial regions within the ischemic hemisphere ofthe stroke-induced rats treated with the U-IGF1R⁺ MSCs.

A cerebral microcirculation of the ischemic area of the stroke-inducedrats is further analyzed by a FITC-dextran perfusion study in thisexample to determine the angiogenesis in the ischemic area of thestroke-induced rats. FIG. 15B shows the analytical results of theFITC-dextran perfusion study in the stroke-induced rats administeredwith the IGF1R⁺ MSCs treatment. The rats are subdivided into threetreatment groups: (1) the stroke-induced rats treated with the U-IGF1R⁺MSCs (n=6), (2) the stroke-induced rats treated with the F-IGF1R⁺ MSCs(n=6), and (3) the stroke-induced rats injected with PBS as controlgroup (n=6). In FIG. 15B, brains perfused with the FITC-dextran showsignificantly enhanced cerebral microvascular perfusion in the U-IGF1R⁺MSCs treatment group compared to the F-IGF1R⁺ MSCs treatment group andthe control group.

Finally, vascular endothelial cell marker CD31 in the ischemic area ofthe stroke-induced rats is labeled by the immunofluorescence stainingassay to determine the blood vessel density. The detected groups includethe stroke-induced rats treated with the U-IGF1R⁺ MSCs (n=6), thestroke-induced rats treated with the F-IGF1R⁺ MSCs (n=6), and thestroke-induced rats injected with the PBS as the control group (n=6).FIG. 15C shows the analytical results of the blood vessel density assaysin the stroke-induced rats administered with the IGF1R⁺ MSCs treatment.In FIG. 15C, the blood vessel density of the U-IGF1R⁺ MSCs treatment ishigher than other groups, wherein p<0.05 when the U-IGF1R⁺ MSCstreatment group compares to the F-IGF1R⁺ MSCs treatment group, andp<0.01 when the U-IGF1R⁺ MSCs treatment group compares to the controlgroup.

These results in this example suggest that the IGF1R⁺ MSCs treatment canimprove the angiogenesis in the ischemic area of the stroke-inducedrats. In particular, the U-IGF1R⁺ MSCs rather than the F-IGF1R⁺ MSCshave superior angiogenesis potential in the ischemic area of the braintissues.

2.6 the IGF1R⁺ MSCs Implantation Facilitates Cerebral Blood Flow (CBF)in the Ischemic Brain

Increased blood vessel density is often associated with an increasedcerebral blood flow (CBF), more efficient delivery of oxygen, nutrients,and enhancing neuronal survival. Therefore, we monitor the CBF in theischemic area of the stroke-induced rats in this example. Thestroke-induced rats are anesthetized by chloral hydrate, and baselinelocal CBF is monitored immediately after cerebral ischemia using a laserdoppler flowmeter (LDF monitor, Moor Instrument). The detected groupsinclude the stroke-induced rats treated with the U-IGF1R⁺ MSCs (n=6),the stroke-induced rats treated with the F-IGF1R⁺ MSCs (n=6), and thestroke-induced rats injected with the PBS as control group (n=6). FIG.16 shows the analytical results of the CBF in the stroke-induced ratsadministered with the IGF1R⁺ MSCs treatment. In FIG. 16, the IGF1R⁺ MSCstreatment can increase the CBF in the ischemic area of thestroke-induced rats. Furthermore, there is significantly more CBF withinthe ischemic area of the stroke-induced rats in the U-IGF1R⁺ MSCstreatment group, wherein p<0.05 when the U-IGF1R⁺ MSCs treatment groupcompares to the F-IGF1R⁺ MSCs treatment group, and p<0.01 when theU-IGF1R⁺ MSCs treatment group compares to the control group.

2.7 Interactions of the CXCR4 and the IGF1R Modulate the IGF1R⁺MSCs-Induced Neurite Regeneration

In order to demonstrate whether the interaction between the IGF1R⁺ MSCsand neural tissue stimulated neurite outgrowth in vivo and in vitro, wequantify neurite regenerations in the ischemic area of thestroke-induced rats and in the MSCs/PCCs (primary cortical culture)co-cultures.

In the in vivo neurite regeneration test, the IGF1R⁺ MSCs areintravenous transplanted into the stroke-induced rats. The test groupsare the U-IGF1R⁺ MSCs transplanted group, the F-IGF1R⁺ MSCs transplantedgroup, the blocking group, and the control group, wherein the blockinggroup are the stroke-induced rats transplanted the U-IGF1R⁺ MSCstransduced with the LV-IGF1R-sh (LV-IGF1R-sh-IGF1R⁺ MSCs) and thestroke-induced rats transplanted the U-IGF1R⁺ MSCs transduced with theLV-CXCR4-sh (LV-CXCR4-sh-IGF1R⁺ MSCs), and the control group is thestroke-induced rats injected with the PBS. The stroke-induced rats aresacrificed at 28 days after transplanting the IGF1R⁺ MSCs, and the braintissue samples are fixed and immunostained with specific antibodyagainst βIII-tubulin. Image anyalysis software (SigmaScan) is used forquantifying length of the neurite, and neurons with processes greaterthan twice the cell body diameter are counted as neurite-bearing cells.

FIG. 17A shows the analytical results of the in vivo neuriteregeneration test. In FIG. 17A, intravenous IGF1R⁺ MSCs transplantationsimprove the neurite regeneration in the ischemic area of thestroke-induced rats. In the U-IGF1R⁺ MSCs transplanted group, the numberof the neurite-bearing cells is significantly higher than other groups,and the neurite length is significantly longer than other groups.However, the results of the blocking groups show that the increase inthe neurite length and the number of the neurite-bearing cells areabolished following treatment with the LV-IGF1R-sh-IGF1R⁺ MSCs andLV-CXCR4-sh-IGF1R⁺ MSCs.

To evaluate whether the IGF1R⁺ MSCs could affect the response of thePCCs to oxygen glucose deprivation (OGD), the neurite regeneration andneuronal survival are measured in PCCs co-cultured with or without theIGF1R⁺ MSCs in the OGD in this example. The IGF1R⁺ MSCs include theU-IGF1R⁺ MSCs, the F-IGF1R⁺ MSCs, and the blocking groups, wherein cellsin the blocking groups are the LV-IGF1R-sh-IGF1R⁺ MSCs and theLV-CXCR4-sh-IGF1R⁺ MSCs. The control group in this example is the PCCsalone cultured in the OGD. The co-cultured cells are fixed andimmunostained with specific antibody against βIII-tubulin, and theneurite regeneration and neuronal survival are measured byaforementioned methods.

FIG. 17B shows the analytical results of the in vitro neuriteregeneration test. In FIG. 17B, the IGF1R⁺ MSCs/PCCs co-culturesfollowing the OGD can increase the neurite length and the number ofneurite-bearing cells. It suggests that the IGF1R⁺ MSCs can improve theneurite regeneration of the PCCs under the OGD. In addition, the neuritelength is significantly longer and more neurite-bearing cells are foundin the U-IGF1R⁺ MSCs/PCCs co-cultures compared to the F-IGF1R⁺ MSCs/PCCsco-cultures and control group. Conversely, the PCCs co-cultures with theLV-IGF1R-sh-IGF1R⁺ MSCs or LV-CXCR4-sh-IGF1R⁺ MSCs show no improvementof the neurite length and the number of neurite-bearing cells.

The results of the in vivo and the in vitro neurite regeneration testindicate that the IGF1R⁺ MSCs can improve the neurite regeneration inthe ischemic and hypoxic brain tissues or brain cells. In particular,the U-IGF1R⁺ MSCs have superior neurite regeneration potential in theischemic and hypoxic brain tissues or brain cells that required theIGF1R and the CXCR4 receptor pathways.

III. The IGF1R⁺ MSCs Used for Treating the Ischemic Heart Disease

The data of the first part examples demonstrate that the IGF1R⁺ MSCshave self-renewal capability and multipotent capability, and the secondpart examples demonstrate that the IGF1R⁺ MSCs can be used for treatingthe brain tissue damage. In the third part examples, we further discussthe effect on the IGF1R⁺ MSCs used for treating the ischemic heartdisease.

3.1 the IGF1R⁺ MSCs Treatment Attenuate the Post-MI LV Dysfunction andReduce the Infarct Size after the MI

In attempting to emphasize that IGF1R⁺ MSCs play a significant role inrescuing the heart from ischemic damage, we assess that in a rat modelof an acute myocardial infarction (AMI) in the third part examples.

The rats are subjected to AMI by ligation of left anterior descending(LAD) coronary artery to simulate transient cardiac ischemia symptoms inthe rats. The test animals are male Sprague-Dawley (SD) rats weighing225-275 g. In brief, after induction of anaesthesia with 2% isofluranein 100% oxygen, the rats receive artificial ventilation using arespirator (SN-480-7) with a tidal volume of 1 mL/100 g and respiratoryrate 80/min. A left thoracotomy is performed in the 4-5th intercostalspace using a rib retractor (MY-9454S), and the left lung is deflatedusing a small piece of gauze soaked in saline. The pericardium is thenremoved and an intra-myocardial ligature places 1-2 mm below theatrioventricular groove using a 6-O polyethylene suture needle withthread (Ethicon). When ligation area becomes white and T-wave of anelectrocardiogram great rise, lungs are then re-inflated before thethorax is closed. The AMI-induced rats are then subdivided into threetreated groups and injected intravenously with MSCs (2×10⁶ cells),IGF1R⁺ MSCs (2×10⁶ cells) or saline as the control group post-MIimmediately. Sham rats undergo the same protocol with the exception ofthe ligation of the coronary artery.

FIG. 18 is a photograph showing a MI area in the AMI-induced ratstreated with IGF1R⁺ MSCs. The AMI-induced rats are sacrificed at 28 daysafter transplanting the IGF1R⁺ MSCs or the MSCs. Heart tissue samplesare soaked in triphenyltetrazolium chloride, and then soaked indehydrogenase, wherein the MI area is stained red-blue, and healthyviable heart muscle is stained deep red. In FIG. 18, the MSCs treatmentand the IGF1R⁺ MSCs treatment can reduce the infarct size and increase athickness of an infarct arterial wall in the AMI-induced rats. Inaddition, the infarct size of the IGF1R⁺ MSC-treated group is muchsmaller than the infarct size in the MSC-treated group and the controlgroup, and the thickness of infarct arterial wall of the IGF1R⁺MSC-treated group is much thicker than the thickness of infarct arterialwall in the MSC-treated group and the control group.

M-mode tracing of echocardiography is further performed for LV functionassessment at 28 days post-MI to determine the effect on the IGF1R⁺ MSCsused for treating the ischemic heart disease in this example. Theassessed groups include the AMI-induced rats treated with the MSCs, theAMI-induced rats treated with the IGF1R⁺ MSCs, the control group, andthe sham rats. FIG. 19A is a transthoracic echocardiogram of theAMI-induced rats treated with the IGF1R⁺ MSCs. FIG. 19B is aquantitative diagram of the transthoracic echocardiogram of theAMI-induced rats treated with the IGF1R⁺ MSCs. In FIGS. 19A and 19B, thetransthoracic echocardiogram results show lower left ventricular endsystolic diameter (LVESD)/left ventricular end diastolic diameter(LVEDD) and higher fraction shortening (FS)/ejection fraction (EF) inthe IGF1R⁺ MSCs treatment group and the MSCs treatment group than thatof the control group at 28 days post-MI, but not influence of the heartrates among the groups. It indicates that the IGF1R⁺ MSCs treatment andthe MSCs treatment can improve cardiac function in the AMI-induced rats.In addition, improvement of the cardiac function of the AMI-induced ratsin the IGF1R⁺ MSCs treatment group is greater than other groups.

3.2 Anti-Inflammatory Effect of the IGF1R⁺ MSCs on Ischemic Myocardium

To further examine whether the MSCs treatment and the IGF1R⁺ MSCstreatment suppress an inflammatory response post-MI in the AMI-inducedrats, we perform an immunohistochemical analysis for studying theinflammatory cell infiltration, the immunofluorescence staining assayfor detecting the expressions of macrophages, and the qRT-PCR fordetecting the expression of various pro-inflammatory factors in theheart tissue of the AMI-induced rats, wherein the AMI-induced rats aresacrificed at 3 days after the MSCs treatment and the IGF1R⁺ MSCstreatment.

FIG. 20 shows the analytical results of the immunohistochemical analysisin the AMI-induced rats at 3 days after the IGF1R⁺ MSCs treatment. InFIG. 20, upper figures are the micrographs of hematoxylin and eosin(H&E) stain in heart tissue slices of the AMI-induced rats. According tothe results of the immunohistochemical analysis, inflammatory conditionsof the heart tissue are categorized into six grades (0, 1, 2, 3, 4, or5), and the higher grade represents more severe inflammation. Theresults taken from different treated groups are classified according tothe above criteria and then gathered statistics. The statistic result isshown in lower figure of the FIG. 20. In FIG. 20, the MSCs treatment andthe IGF1R⁺ MSCs treatment reduce the inflammation in the heart tissuesof the AMI-induced rats. Furthermore, significant reduction of theinflammation is observed in the IGF1R⁺ MSCs treatment group compared tothe MSCs treatment and control group, wherein p<0.05 when the MSCstreatment group compares to the control group, and p<0.01 when theIGF1R⁺ MSCs treatment group compares to the control group.

To further detect the expression of the macrophages in the AMI-inducedrats treated with the MSCs or the IGF1R⁺ MSCs at 3 days after the MSCstreatment and the IGF1R⁺ MSCs treatment, we perform theimmunofluorescence staining assay labeling a macrophage marker CD68 inthe heart tissue slice of the AMI-induced rats, and the results areobserved by using a fluorescent microscope. FIG. 21 shows the analyticalresults of the immunofluorescence staining assay in the AMI-induced ratsat 3 days after the IGF1R⁺ MSCs treatment or the MSCs treatment. In FIG.21, fewer CD68⁺ cells are infiltrated at the peri-infarct area at 3 daysafter the MI in the MSC treatment group or the IGF1R⁺ MSC treatmentgroup than that of the control group. In addition, the IGF1R⁺ MSCtreatment group shows significantly less expression of the CD68⁺ cellsthan the MSCs treatment group and the control group. It indicates thatthe IGF1R⁺ MSC treatment can greatly improve the inflammatory conditionin the heart tissue of the AMI-induced rats.

Increased expression of proinflammatory cytokines are usually observedwith many inflammatory cells infiltration in the ischemic myocardium.Quantitative RT-PCR is performed for assessing expressions of variouspro-inflammatory factors in the AMI-induced rats at 3 days post-MI. Thedetected pro-inflammatory factors are IL-1β, IL-6, TNF-α, and INF-γ. Theexpression of anti-inflammatory factor IL-10 is also detected in thisexample. FIG. 22 shows the analytical results of the expression ofpro-inflammatory factors in the AMI-induced rats at 3 days after theIGF1R⁺ MSCs treatment. In FIG. 22, the expression of thepro-inflammatory factors of IL-1β, IL-6, TNF-α, and INF-γ are decreasedin the heart tissues at 3 days post-MI of the AMI-induced rats treatedwith the MSCs or the IGF1R⁺ MSCs. In particular, mRNA expression levelsof the pro-inflammatory factors of IL-1β, IL-6, TNF-α, and INF-γ aresignificantly reduced in the IGF1R⁺ MSC treatment group compared to theMSCs treatment group and the control group. In contrast, the expressionof the anti-inflammatory factor IL-10 is increased in the MSCs treatmentgroup and the IGF1R⁺ MSCs treatment group. In particular, significantincrease level of IL-10 is found in the IGF1R⁺ MSC treatment group. Itsuggests that the IGF1R⁺ MSC treatment can greatly improve theinflammatory condition in the heart tissue of the AMI-induced rats.

3.3 the IGF1R⁺ MSCs Treatment Attenuate the MI-Induced Fibrosis

Myocardial fibers are different from muscle fibers, the myocardialfibers can work long hours to pump blood into every part of a body. Themyocardial fibers will produce irreversible necrosis after the MI. Thenecrosis part is replaced with fibrous tissue, and then fibrosis isgenerated in a few weeks. To examine whether the IGF1R⁺ MSCs treatmentcould attenuate the MI-induced fibrosis, we perform a Masson's trichromestain for studying fibrosis conditions in the heart tissue of theAMI-induced rats, wherein the AMI-induced rats are sacrificed at 28 daysafter the MSCs treatment and the IGF1R⁺ MSCs treatment. In the Masson'strichrome stain, collagen fibers are stained blue, and muscle fibers arestained red. The analyzed groups include the AMI-induced rats treatedwith the MSCs, the AMI-induced rats treated with the IGF1R⁺ MSCs, thecontrol group, and the sham rats.

FIG. 23 shows the analytical results of the Masson's trichrome stain inthe AMI-induced rats at 28 days after the IGF1R⁺ MSCs treatment. In FIG.23, the upper figures are the micrographs of the Masson's trichromestain in the heart tissue slices of the AMI-induced rats. According tothe results of the Masson's trichrome stain, the fibrosis conditions ofthe heart tissue are categorized into six grades (0, 1, 2, 3, 4, or 5),and the higher grade represents more severe fibrosis. The results takenfrom different treated groups are classified according to the abovecriteria and then gathered statistics. The statistic result is shown inthe lower figure of the FIG. 23. In FIG. 23, the MSCs treatment and theIGF1R⁺ MSCs treatment attenuate the fibrosis in the heart tissues of theAMI-induced rats. In particular, significantly reduced MI-inducedfibrosis is observed in the IGF1R⁺ MSCs treatment group compared to theMSCs treatment group and the control group, wherein p<0.05 when the MSCstreatment group compares to the control group, and p<0.01 when theIGF1R⁺ MSCs treatment group compares to the control group.

IV. The IGF1R⁺ MSCs Used for Treating a Multiple Sclerosis

The data of the first part examples demonstrate that the IGF1R⁺ MSCshave self-renewal capability and multipotent capability. In the fourthpart examples, we further discuss the effect on the IGF1R⁺ MSCs used fortreating the multiple sclerosis.

4.1 the IGF1R⁺ MSCs Transplantation Improved Neurological Behavior inExperimental Autoimmune Encephalomyelitis Model

In vivo self-renewal and neuroregenerative potentials of the IGF1R⁺ MSCsare assessed in the experimental autoimmune encephalomyelitis mousemodel in the fourth part examples. Mice before and after the multiplesclerosis are measured by neurological deficits modality to evaluate theneurological recovery.

The experimental autoimmune encephalomyelitis (EAE) is used as anexperimental animal model of the multiple sclerosis. Test animals arefemale C57BL/6 mice 6-8 weeks old. The C57BL/6 mice are immunized twice,at day 0 and day 7, by subcutaneous injection with anemulsion-containing purified myelin oligodendrocyte glycoprotein (MOG)peptide 3.7 mg/kg or 15 mg/kg respectively in complete Freund's adjuvant(Difco) containing 200 μg heat-activated Mycobacterium tuberculosis in atotal volume of 0.2 ml. The MOG peptide corresponds to residues 35-55,and the sequence of the MOG peptide is MEVGWYRSPFSRVVHLYRNGK (Sigma).The C57BL/6 mice are also injected with Bordetella pertussis toxin (15μg/kg, Sigma) at day 0 and day 2. Clinical signs of EAE appear typicallyafter 12-14 days, reaching to the maximum score after 5-6 days with norecovery.

At 14 days post-EAE induction, the C57BL/6 mice are subdivided intothree treatment groups and injected with 3 different cells or vehiclefor cell therapy. The cells injected in group 1 are U-IGF1R⁺ MSCs, andthe cells injected in group 2 are U-MSCs, which are unscreened. TheC57BL/6 mice in group 3 are injected with PBS (vehicle) as a controlgroup. The C57BL/6 mice in group 1 and group 2 receive intravenousinjections of 1×10⁶ U-IGF1R⁺ MSCs or U-MSCs on day 10, 15, or 24 afterimmunization. The C57BL/6 mice in group 3 receive PBS in a volume of 50μl through femoral vein. All of the C57BL/6 mice are examined daily forclinical disease severity daily by using a score scale: 0, no diseasesigns; 1, loss of tail tonicity; 2, mild hindlimb weakness; 3, completehindlimb paralysis; 4, paralysis of four limbs; 5, moribund; and 6,death.

FIG. 24 is a graph showing the improvement of neurological behavior inexperimental autoimmune encephalitis-induced mice after the celltreatment. In group 3 injected with PBS alone, symptoms of multiplesclerosis began to appear on day 10 after immunization, and reached thepeak of disease on the day 15 after immunization (the mean clinicalscore reached the highest value of 4). In group 2 administered U-MSCsthree times on the day 10, day 15, and day 24 after immunization, themean clinical score is reduced to about 2 from day 15 to day 60 afterimmunization. There is a statistically significant difference (p<0.01)compared with group 3. In group 1 administered U-IGF1R⁺ MSCs three timeson the day 10, day 15, and day 24 after immunization, the mean clinicalscore from day 15 to day 60 is significantly reduced to approximately 1after immunization. There is a statistically significant difference(p<0.01) compared with group 1, and there is also a statisticallysignificant difference (p<0.01) compared with the group 2. The resultindicates that U-IGF1R⁺ MSCs have excellent therapeutic effects ofmultiple sclerosis, and cell therapy of U-IGF1R⁺ MSCs can be safely andeffectively performed by the intravenous injection.

To sum up, the present disclosure provides the mesenchymal stem cellexpresses the IGF1R on its cell surface (IGF1R⁺ MSC). The IGF1R⁺ MSC hasself-renewal capability and multipotent differentiation capability. Inthe method for the clonogenic expansion of a plurality of IGF1R⁺ MSCs ofthe present disclosure, the culture medium is containing the hUCS,wherein the hUCS is rich in growth factors, especially the PDGF-BB. Thegrowth factors can enhance the expression of the IGF1R in the IGF1R⁺ MSCand maintain the multipotent differentiation capability of the IGF1R⁺MSC. Using the medium containing the hUCS has advantages of easyobtaining easy and avoiding the risk of allergic reactions andinfections of viruses or pathogens resulting from using non-human serum.The method for obtaining a plurality of multipotent MSCs of the presentdisclosure can screen the IGF1R positive cells, or further screen theIL22RA1 positive cells from the mammalian tissue cell mixture, whereinthe screened cells are the MSCs having multipotent differentiationcapability. Therefore, the method can quickly and specifically screenthe multipotent MSCs. Furthermore, the IGF1R⁺ MSC of the presentdisclosure can be used in the cell treatment for treating the ischemicheart disease, the brain tissue damage and the multiple sclerosis. Inmore details, for treating the brain tissue damage, the IGF1R⁺ MSCs canincrease the glucose metabolic activity of the subject, enhance theangiogenesis of the subject, and augments the neurite regeneration ofthe subject. The IGF1R⁺ MSCs have the neuroplasticity effect through theIGF1R and the CXCR4 interactions. For treating the ischemic heartdisease, the IGF1R⁺ MSCs can attenuate the post-MI LV dysfunction,reduce the infarct size after the MI, reduce the fibrosis caused by theMI, and reduce the inflammatory effect on the MI. For treating themultiple sclerosis, the IGF1R⁺ MSCs can significantly improve theneurological behavior of in experimental autoimmune encephalitis-inducedmice.

Although the present disclosure has been described in considerabledetail with reference to certain embodiments thereof, other embodimentsare possible. Therefore, the spirit and scope of the appended claimsshould not be limited to the description of the embodiments containedherein.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the structure of the presentdisclosure without departing from the scope or spirit of the disclosure.In view of the foregoing, it is intended that the present disclosurecover modifications and variations of this disclosure provided they fallwithin the scope of the following claims.

What is claimed is:
 1. A method for treating a multiple sclerosis, themethod comprising administering a composition containing a plurality ofmesenchymal stem cells to a subject in need for a treatment of themultiple sclerosis, wherein each of the mesenchymal stem cells expressesinsulin-like growth factor 1 receptors.
 2. The method of claim 1,wherein the mesenchymal stem cells is obtained by culturing in mediacontaining human cord blood serum.
 3. The method of claim 2, wherein aconcentration of the human cord blood serum in the media is 1-10% (v/v).4. The method of claim 2, wherein the human cord blood serum comprisesplatelet-derived growth factor BB (PDGF-BB).
 5. The method of claim 1,wherein the mesenchymal stem cells are umbilical cord mesenchymal stemcells.
 6. The method of claim 1, wherein the multiple sclerosis is arelapsing-remitting multiple sclerosis (RRMS).
 7. The method of claim 1,wherein the multiple sclerosis is a primary progressive multiplesclerosis (PPMS).
 8. The method of claim 1, wherein the multiplesclerosis is a secondary progressive multiple sclerosis (SPMS).
 9. Themethod of claim 1, wherein the multiple sclerosis is a progressiverelapsing multiple sclerosis (PRMS).
 10. The method of claim 1, whereinthe composition is administered by an intravenous injection.